Characterization of novel virulence factors of Shigella flexneri and development of Caenorhabditis elegans as an animal model for Shigella infection

Abstract

The Gram-negative bacterium Shigella flexneri is the causative agent of Shigellosis, also known as bacillary dysentery, responsible for 5-15% of the global burden of diarrhoeal disease. Despite over 50 years of Shigellosis research, the search for a commercially viable vaccine is ongoing. A thorough understanding of bacterial pathogenesis is paramount for the development of an effective vaccine. To this end the aim of this thesis is to further our understanding of S. flexneri pathogenesis by characterizing novel virulence factors and to develop a new animal model of shigellosis. The first aim of this study is to investigate the role of L-asparaginase (AnsB) and gamma-glutamyltranspeptidase (GGT) in S. flexneri pathogenesis. Using a reverse genetic approach I found that AnsB and GGT are required for bacterial adherence to host cells. In vivo studies in both the Caenorhabditis elegans and the murine pulmonary model of shigellosis revealed that AnsB and GGT contribute to S. flexneri virulence. Differential in-gel electrophoresis (DIGE) showed that ansB and ggt mutations exert pleiotropic effects on the expression of a number of S. flexneri genes, including prominent bacterial outer membrane proteins, OmpA and YaeT. This is the first report in S. flexneri where the functions of AnsB and GGT have been found to extend beyond their canonical metabolic roles. The requirement of AnsB and GGT for the virulence of S. flexneri makes these genes attractive candidates for designing new Shigella vaccine strategies. The second aim of this study is to identify bacteriophage genes that contribute to S. flexneri virulence. To date in S. flexneri, the O-antigen modifying genes are the only bacteriophage genes that have been linked to host virulence. Here the S. flexneri phage SfII was isolated from a highly prevalent S. flexneri serotype 2a strain and completely sequenced to identify novel bacteriophage-encoded genes. In parallel, uncharacterized genes in the S. flexneri phage, SfV were studied to identify potential phage-encoded virulence factors. A mutant lysogenic strain lacking five phage genes identified as expressed in the host, orf28-32, was generated. In vitro and in vivo virulence studies indicate that genes within the SfV orf28-32 cluster play a role in host virulence. This is the first reported study to identify bacteriophage-encoded virulence factors outside of the O-antigen modifying cluster in S. flexneri. The third aim of this study is to characterize C. elegans as a small animal model of shigellosis. In recent years the use of C. elegans as an animal model for several microbial diseases has been gaining momentum. Using electron microscopy, I have shown that virulent strains of S. flexneri are ingested by C. elegans and that S. flexneri cells invade the nematode intestinal cells. DIGE was used to compare the proteomes of nematodes infected with S. flexneri and control strains to successfully identify nematode responses to S. flexneri. These findings are significant as they provide further evidence supporting the use of C. elegans as a viable model to study shigellosis. Show more